The Cellvibrio japonicus Mannanase CjMan26C Displays a Unique exo-Mode of Action That Is Conferred by Subtle Changes to the Distal Region of the Active Site

Abstract

The microbial degradation of the plant cell wall is a pivotal biological process that is of increasing industrial significance. One of the major plant structural polysaccharides is mannan, a ␤-1,4-linked D-mannose polymer, which is hydrolyzed by endoand exo-acting mannanases. The mechanisms by which the exoacting enzymes target the chain ends of mannan and how galactose decorations influence activity are poorly understood. Here we report the crystal structure and biochemical properties of CjMan26C, a Cellvibrio japonicus GH26 mannanase. The exoacting enzyme releases the disaccharide mannobiose from the nonreducing end of mannan and mannooligosaccharides, harnessing four mannose-binding subsites extending from ؊2 to ؉2. The structure of CjMan26C is very similar to that of the endo-acting C. japonicus mannanase CjMan26A. The exo-activity displayed by CjMan26C, however, reflects a subtle change in surface topography in which a four-residue extension of surface loop creates a steric block at the distal glycone ؊2 subsite. endoActivity can be introduced into enzyme variants through truncation of an aspartate side chain, a component of a surface loop, or by removing both the aspartate and its flanking residues. The structure of catalytically competent CjMan26C, in complex with a decorated manno-oligosaccharide, reveals a predominantly unhydrolyzed substrate in an approximate 1 S 5 conformation. The complex structure helps to explain how the substrate "side chain" decorations greatly reduce the activity of the enzyme; the galactose side chain at the ؊1 subsite makes polar interactions with the aglycone mannose, possibly leading to suboptimal binding and impaired leaving group departure. This report reveals how subtle differences in the loops surrounding the active site of a glycoside hydrolase can lead to a change in the mode of action of the enzyme.The plant cell wall represents the dominant source of organic carbon in the biosphere and as such supports many facets of terrestrial and marine life. Access to this valuable source of carbon is mediated by extensive microbial enzyme consortia, which generate sugars that are utilized by microbial ecosystems to the benefit of plants, mammalian herbivores, and insects. These degradative enzymes are increasingly deployed in industrial processes, and it is apparent that their use in producing second generation, lignocellulosebased, biofuels is of considerable environmental significance (1, 2). Among the major polysaccharides in softwoods and angiosperms are the mannans. These polysaccharides comprise a backbone of ␤-1,4-linked D-mannopyranose sugars (known as "mannan") or a heterogeneous combination of ␤-1,4-D-mannose and ␤-1,4-D-glucose units (termed "glucomannan"), which can be decorated with ␣-1,6-galactose side chains to yield "galactomannan" and "galactoglucomannan," respectively (3). For complete enzymatic degradation, the galactose decorations are removed by ␣-galactosidases (4, 5), whereas the internal glycosidic linkages in mannans are cleaved by endo-␤-1,4 mannanases ...

The Biochemistry and Structural Biology of Plant Cell Wall Deconstruction

“…This article will not discuss these GH16 enzymes, which are covered in detail in the review by Eklöf and Brumer (2010;this issue). An unusual feature of b-mannanases is that substrate specificity is not conferred by the recognition of Man in its relaxed chair conformation ( 4 C 1 ) at the critical 21 subsite (glycosidic bond cleavage occurs between the sugars bound at the 21 and +1 subsites; Davies et al, 1997) but through the B 2,5 topology displayed by the oxocarbonium transition state (Ducros et al, 2002;Tailford et al, 2007;Cartmell et al, 2008).…”

“…Indeed, a cohort of GH26 mannanases contain an Arg at the 22 subsite that makes extensive interactions with the substrate and appears to confer unusually high activity against small mannooligosaccharides (Ducros et al, 2002;Cartmell et al, 2008). Screening genomic databases for other GH26 enzymes that retain this Arg may facilitate the identification of novel mannooligosaccharidases.…”

“…Screening genomic databases for other GH26 enzymes that retain this Arg may facilitate the identification of novel mannooligosaccharidases. Currently, the two Cellvibrio enzymes that contain a highaffinity 22 subsite do not possess additional negative binding subsites, which may explain why the high activity displayed against mannotriose and mannotetraose is not translated to the hydrolysis of polysaccharides (Hogg et al, 2001;Cartmell et al, 2008).…”

“…Thus, small loop extensions surrounding the distal subsite that accommodates the nonreducing end of the substrate create steric constraints that prevent extension of the substrate beyond this subsite. Variants of these enzymes, in which the loop extensions have been removed, display an endo mode of action (Proctor et al, 2005;Cartmell et al, 2008;Ochiai et al, 2009;Fig. 2).…”

Introducing endo-xylanase activity into an exo-acting arabinofuranosidase that targets side chains

“…Thus, endo-acting glycoside hydrolases, which cleave internal linkages, display a substrate binding cleft open at both ends, whereas the active site of exo-acting enzymes that target the termini of polysaccharide backbones display a substrate binding groove blocked at one end by extended loops. Endo and backbone exo-acting enzymes are closely related; in nature, short loop extensions have led to the conversion of endo-glycanases into exo-glycanases that target the polysaccharide backbone (6)(7)(8). The active site of enzymes that cleave sugars decorating the polysaccharide backbone displays a pocket topology in which the target side chain is positioned (9,10).…”